Calicheamicin γ1 is a naturally occurring antibiotic and one of the most potent cellular toxins known (Figure 8.19). A single molecule of calicheamicin γ1, after entering a cell and cleaving a covalent bond in DNA, is capable of causing cell death. How is this possible? In Chapter 6 we have seen how some electrophilic chemotherapy drugs alkylate and cross-link strands of DNA. In contrast, calicheamicin causes breaks in the DNA strand employing a radical-mediated reaction. In a series of choreographed events, calicheamicin first binds to DNA and is then reduced and undergoes multiple structural rearrangements leading to a diradical species that attacks DNA. In this case study we will discuss some of the key reactions involved in this process.
Figure 8.19 Structure of calicheamicin γ1.
The chemical structure of calicheamicin is remarkable and reflects the complexity of its mechanism of action. The highly substituted aryl ring and four sugars in the molecule are mainly responsible for specificity in binding of the molecule to the minor grove of DNA. The highly unsaturated enediyne function (an alkene flanked by two alkynes) is the “warhead” of calicheamicin, a chemical precursor to the diradical species that will ultimately do irreversible damage to DNA. If the enediyne is the warhead, then the three sulfur atoms and the electrophilic Michael acceptor together comprise a “trigger” that must be pulled to unleash the warhead.
After binding in the minor groove of DNA, a disulfide bond in calicheamicin is reduced in an enzymatic process to yield a nucleophilic thiol side chain (Figure 8.20). The thiol undergoes intramolecular Michael addition to the cyclohexenone ring that is part of the bridged bicyclic ring system. An important consequence of this Michael reaction is that the two alkynes of the enediyne are brought slightly closer together in space. This in turn leads to an electrocyclization reaction (Bergman cyclization) that produces an aryl diradical intermediate.
Figure 8.20 Reaction sequence leading to activation of the calicheamicin enediyne and Bergman cyclization to produce an aryl diradical intermediate. For clarity, the sugar moiety is not shown.
The aryl diradical is the species that abstracts a hydrogen atom from the backbone of DNA. This can happen at various sites, one of which is the 5′ carbon directly adjacent to phosphate in the DNA backbone (Figure 8.21). Once generated, the 5′ carbon radical reacts with oxygen to form a peroxy radical and, after abstracting a hydrogen atom, a hydroperoxide intermediate. Note that these steps are similar to the propagation steps involved in the oxidation of linoleic acid (Figure 8.12). The DNA strand is broken via breakdown of the 5′ hydroperoxide acetal, with phosphate serving as the (excellent) leaving group. This is but one mechanism by which the calicheamicin diradical can cleave DNA; strand breaks can also occur following hydrogen abstraction from the 1′, 3′, or 4′ carbons. In each case the cell is unable to repair the strand breaks and this leads to cell death.
Figure 8.21 The radical-mediated cleavage of DNA, initiated by reaction with the activated, diradical form of calicheamicin (the letter B represents the nucleoside bases of the DNA strand).
These potent cell-killing effects generated interest in using calicheamicin γ1 to treat cancer. Not surprisingly, the compound was found to exhibit little selectivity between cancer cells and normal cells. However, by attaching calicheamicin γ1 to an antibody that specifically binds leukemia cells, the antibody-drug conjugate (ADC) Mylotarg was produced. Mylotarg was used from 2000 to 2010, but concerns over its safety and efficacy led to its voluntary withdrawal from the U.S. market in 2010. Nonetheless, ADC therapies continue to attract great interest in oncology, with ~40 agents in clinical trials as of May 2015.
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